Donor repair templates multiplex genome editing

Inventors

LEE, Baeckseung • ASTRAKHAN, Alexander

Assignees

Novo Nordisk AS

Abstract

The present disclosure provides improved multiplex genome editing compositions and methods. The disclosure further provides genome edited cells for the prevention, treatment, or amelioration of at least one symptom of a hemoglobinopathy, a cancer, an infectious disease, an autoimmune disease, an inflammatory disease, or an immunodeficiency.

Core Innovation

The invention relates to editing the genome of a cell at multiple target sites using one or more engineered nucleases and a DNA donor repair template. The DNA donor repair template includes from 5′ to 3′ a 5′ homology arm homologous to a DNA sequence 5′ of a first target site, a 5′ homology arm homologous to a DNA sequence 5′ of a second target site, a transgene, a 3′ homology arm homologous to a DNA sequence 3′ of the second target site, and a 3′ homology arm homologous to a DNA sequence 3′ of the first target site.

This arrangement provides paired homology-arm regions spanning across two target sites while carrying a transgene payload. The method uses engineered nucleases together with DNA donor repair template-mediated genome editing at two target sites, in a single donor repair template that contains homology arms associated with both the first and second target sites and includes a transgene within the same donor molecule.

The description characterizes immune-related target genes in immune effector and hematopoietic cells, including TCRα and immune system checkpoint genes or genes encoding immunosuppressive signaling components. Example target categories include TCR signaling components, checkpoint genes such as PD-1, LAG-3, TIM-3, CTLA-4, BTLA, TIGIT, VISTA, and KIR, and genes encoding immunosuppressive signaling components such as IL-10Rα, TGFβR1, TGFβR2, AHR, SGK1, TSC2, VHL, A2AR, and CBLB.

Transgene examples described include engineered antigen receptors, including CAR and engineered TCR, as well as immunopotency enhancers such as BiTE and cytokines, immunosuppressive signal dampers, and flip receptors such as PD-1 flip receptor, LAG-3 flip receptor, TIM-3 flip receptor, CTLA-4 flip receptor, TIGIT flip receptor, and TGFβRII flip receptor.

Claims Coverage

Independent inventive coverage is provided by one independent method claim, describing multiplex genome editing at two target sites using engineered nucleases and a single DNA donor repair template containing a transgene flanked by 5′ and 3′ homology arms arranged across the two target sites.

The independent claim centers on a multiplex editing donor design: a single DNA donor repair template with 5′ and 3′ homology arms corresponding to both a first and a second target site, together with a transgene, used in combination with one or more engineered nucleases to edit a cell's genome at multiple target sites.

Stated Advantages

Documented Applications